Molecular Mechanics of FHC and DCM Mutant Actomyosin

University Of Vermont & St Agric College, Burlington VT

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Abstract

The goal of this proposal is to provide a molecular basis for the clinical impact that point mutations to cardiac actin and myosin's molecular structure have on patients afflicted with familial hypertrophic (FHC) and dilated cardiomyopathy (DCM). FHC is characterized by a thick, hypercontractile ventricular wall, whereas DCM patients have ventricles that are thin and hypocontractile. To understand how point mutations within actin or myosin result in such drastically different pathologies, information about the molecular mechanics of the mutant actomyosin motor is required. Our approach will take advantage of both transgenic mice and the Baculovirus system to express mutant cardiac actin and myosin that have single amino acid substitutions found in either FHC or DCM. Using state-of-the-art laser trapping techniques in an in vitro motility assay in force clamp mode, we will measure the force:velocity relationship of a small myosin ensemble (<50 molecules) as it interacts with a single actin or regulated thin filament. These data will provide an estimate of the maximum power that can be produced by a mutant actomyosin motor. Any alterations in power will be probed at the level of a single myosin molecule to determine if changes have occurred to the inherent motion generating capacity or to the rates of actomyosin transitions. Since the chosen mutations are localized throughout the myosin heavy chain (FHC: R403Q, R453C, G741R; DCM: S532P, F764L), we will directly pinpoint crucial intramolecular domains that are important to myosin's ability to generate force and motion. With actin being a key element in force production and thin filament regulation, mutations to actin that lead to FHC (E99K, A331P) and DCM (R312H, E361G) will be assessed for their impact on actin flexural rigidity, actomyosin power production, and thin filament regulation. These studies will help determine whether the impact of these point mutations on actomyosin's mechanical performance results in functional alterations that are common and distinct for a given form of hypertrophy so that one triggers a cascade of events resulting in either FHC or DCM.